Lactate Efflux Research Articles

3-Nitropropionic acid induces cell death and mitochondrial dysfunction in rat corticostriatal slice cultures

Exposure of organotypic rat corticostriatal slice cultures to the mitochondrial toxin 3-nitropropionic acid (3-NP) resulted in concentration-dependent loss of cresylviolet-stained cells and increase of lactate dehydrogenase and lactate efflux into the culture medium, indicators for cell death and metabolic activity in the slices, respectively. The involvement of apoptosis in these slices was suggested by using the terminal transferase-mediated biotinylated-UTP nick end-labeling (TUNEL) technique, and immunohistochemistry for the apoptosis-related markers Bax and Bcl-2. In 3-NP-exposed slices, TUNEL-positive cells were observed in both the striatum and the cortex but in different forms: striatal neurons were either diffusely stained or showed nuclear fragmentation, cortical neurons only exhibiting nuclear fragmentation. In 3-NP-exposed slices, the pro-apoptotic protein Bax was abundantly expressed, whereas the anti-apoptotic protein Bcl-2 was not expressed in striatal neurons. We suggest that both apoptosis and necrosis are involved in the 3-NP-treated slices, apoptosis as well as necrosis in the striatum and apoptosis in the cortex.

Recovery of trout myocardial function following anoxia: preconditioning in a non-mammalian model.

Studies with mammals and birds clearly demonstrate that brief preexposure to oxygen deprivation can protect the myocardium from damage normally associated with a subsequent prolonged hypoxic/ischemic episode. However, is not known whether this potent mechanism of myocardial protection, termed preconditioning, exists in other vertebrates including fishes. In this study, we used an in situ trout (Oncorhynchus mykiss) working heart preparation at 10 degrees C to examine whether prior exposure to 5 min of anoxia (PO(2) < or = 5 mmHg) could reduce or eliminate the myocardial dysfunction that normally follows 15 min of anoxic exposure. Hearts were exposed either to a control treatment (oxygenated perfusion) or to one of three anoxic treatments: 1) anoxia with low P(out) [15 min of anoxia at an output pressure (P(out)) of 10 cmH(2)O]; 2) anoxia with high P(out) [10 min of anoxia at a P(out) of 10 cmH(2)O, followed by 5 min of anoxia at P(out) = 50 cmH(2)O]; and 3) preconditioning [5 min of anoxia at P(out) = 10 cmH(2)O, followed after 20 min of oxygenated perfusion by the protocol described for the anoxia with high P(out) group]. Changes in maximum cardiac function, measured before and after anoxic exposure, were used to assess myocardial damage. Maximum cardiac performance of the control group was unaffected by the experimental protocol, whereas 15 min of anoxia at low P(out) decreased maximum stroke volume (V(s max)) by 15% and maximum cardiac output (Q(max)) by 23%. When the anoxic workload was increased by raising P(out) to 50 cmH(2)O, these parameters were decreased further (by 23 and 38%, respectively). Preconditioning with anoxia completely prevented the reductions in V(s max) and Q(max) that were observed in the anoxia with high P(out) group and any anoxia-related increases in the input pressure (P(in)) required to maintain resting Q (16 ml. min(-1). kg(-1)). Myocardial levels of glycogen and lactate were not affected by any of the experimental treatments; however, lactate efflux was sevenfold higher in the preconditioned hearts. These data strongly suggest that 1) a preconditioning-like mechanism exists in the rainbow trout heart, 2) increased anaerobic glycolysis, fueled by exogenous glucose, was associated with anoxic preconditioning, and 3) preconditioning represents a fundamental mechanism of cardioprotection that appeared early in the evolution of vertebrates.

Lactate efflux and the neuroenergetic basis of brain function.

In the unstimulated brain energy is primarily supplied by the oxidation of glucose. However the oxygen-to-glucose index (OGI), which is the ratio of metabolic rates of oxygen to glucose, CMR(O2)/CMR(glc), diverges from the theoretical value of 6 as activity is increased. In vivo measurements of brain lactate show its concentration to increase with stimulation. The decreasing OGI with stimulation had led to the suggestion that activation, unlike resting activity, is supported by anaerobic glycolysis. To date a unifying concept that accommodates glucose oxidation at rest with lactate generation and OGI decrease during stimulation of brain is lacking. Furthermore, energetics that change with increasing activity are not consistent with a neuroenergetic model that has been proposed from 1-(13)C-glucose MRS experiments. That model, based upon in vivo MRS measurements and cellular studies by Pellerin and Magistretti, showed that glutamate neurotransmitter cycling was coupled to glucose oxidation over a wide range of brain activities from rest down to deep anesthesia. Here we reconcile these paradoxical observations by suggesting that anaerobic glucose consumption (which can provide energy rapidly) increases with activation to meet the power requirements of millisecond neuronal firing. It is proposed, in accord with our neuroenergetic model, that the extra glucose mobilized rapidly for glial clearance of glutamate, is not needed for the oxidative processes that are responsible for neuronal firing and glutamate release, and consequently it is effluxed as lactate. A stoichiometric relation between OGI and lactate concentration is derived from the neuroenergetic model, showing that the enhanced glucose uptake during activation is consistent with neuronal activity being energetically supported by glucose oxidation.

Opioid preconditioning: myocardial function and energy metabolism

Background. Opioid receptor agonists are involved in ischemic preconditioning and natural hibernation. The aim of this study was to determine whether pretreatment with d-Ala2-Leu5-enkephalin or morphine confers cardioprotection in large mammalian hearts. We assessed myocardial functional recovery and global energy metabolism after ischemic cold storage. Methods. After pretreatment with d-Ala2-Leu5-enkephalin, morphine sulfate, or saline (n = 6 each), swine hearts were excised and stored for 75 minutes at 4°C, then reperfused in a four-chamber isolated working heart apparatus. Serial myocardial biopsies were performed to assess cellular energy metabolism. Results. Improved systolic (cardiac output, contractility) and diastolic (tau) left ventricular functions were observed in hearts pretreated with d-Ala2-Leu5-enkephalin or morphine. These benefits were not correlated with changes in high-energy phosphate levels. Cardiac enzyme leakage (creatine kinase, troponin-I) was similar among treated and control groups. Lactate efflux increased significantly in controls, but not in opioid-pretreated hearts ( p < 0.01) at 75 minutes of reperfusion. Conclusions. d-Ala2-Leu5-enkephalin and morphine pretreatments improve postischemic function after cold storage of swine hearts. Postischemic lactate reduction, but not high-energy phosphate levels, may account for the observed cardioprotective effects.

Calcitonin (CT) rapidly increases NA(+)/H(+) exchange and metabolic acid production: effects mediated selectively by the C1A CT receptor isoform.

Two isoforms of the calcitonin receptor are expressed in rabbit: the common C1a isoform and the calcitonin receptor Delta e13 isoform, which has a deletion in the seventh transmembrane domain. Using microphysiometry, we investigated the effects of calcitonin on proton efflux from HEK293 cells stably transfected with C1a, calcitonin receptor Delta e13, or empty vector. In C1a-expressing cells only, calcitonin rapidly induced a biphasic elevation in proton efflux consisting of an initial transient and a sustained plateau, accompanied by an increase in lactate efflux. Inhibitors of Na(+)/H(+) exchange abolished only the initial transient, whereas removal of extracellular glucose abolished only the sustained plateau. These data suggest that activation of Na(+)/H(+) exchange mediates the initial transient, whereas increased glucose metabolism underlies the sustained plateau. Because both receptor isoforms activate adenylyl cyclase, the lack of effect of calcitonin on proton efflux from calcitonin receptor Delta e13-expressing cells argued against involvement of cAMP in activating proton efflux. Similarly, studies involving elevation or buffering of cytosolic free Ca(2+) concentration argued against involvement of Ca(2+). Activation of PKC mimicked the plateau phase of calcitonin-induced proton efflux from C1a cells, whereas inhibition or depletion of PKC suppressed it. Activation of proton transport and production are novel cellular responses to calcitonin, mediated selectively by the C1a receptor isoform via a mechanism involving PKC.

Evidence of separate pathways for lactate uptake and release by the perfused rat heart.

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.

Extracellular carbonic anhydrase activity facilitates lactic acid transport in rat skeletal muscle fibres.

1. In skeletal muscle an extracellular sarcolemmal carbonic anhydrase (CA) has been demonstrated. We speculate that this CA accelerates the interstitial CO2/HCO3- buffer system so that H+ ions can be rapidly delivered or buffered in the interstitial fluid. Because > 80 % of the lactate which crosses the sarcolemmal membrane is transported by the H+-lactate cotransporter, we examined the contributions of extracellular and intracellular CA to lactic acid transport, using ion-selective microelectrodes for measurements of intracellular pH (pHi) and fibre surface pH (pHs) in rat extensor digitorum longus (EDL) and soleus fibres. 2. Muscle fibres were exposed to 20 mM sodium lactate in the absence and presence of the CA inhibitors benzolamide (BZ), acetazolamide (AZ), chlorzolamide (CZ) and ethoxzolamide (EZ). The initial slopes (dpHs/dt, dpHi/dt) and the amplitudes (DeltapHs, DeltapHi) of pH changes were quantified. From dpHi/dt, DeltapHi and the total buffer factor (BFtot) the lactate fluxes (mM min-1) and intracellular lactate concentrations ([lactate]i) were estimated. 3. BFtot was obtained as the sum of the non-HCO3- buffer factor (BFnon-HCO3) and the HCO3- buffer factor (BFHCO3). BFnon-HCO3 was 35 +/- 4 mM pH-1 for the EDL (n = 14) and 86 /- 16 mM pH-1 for the soleus (n = 14). 4. In soleus, 10 mM cinnamate inhibited lactate influx by 44 % and efflux by 30 %; in EDL, it inhibited lactate influx by 37 % and efflux by 20 %. Cinnamate decreased [lactate]i, in soleus by 36 % and in EDL by 45 %. In soleus, 1 mM DIDS reduced lactate influx by 18 % and efflux by 16 %. In EDL, DIDS lowered the influx by 27 % but had almost no effect on efflux. DIDS reduced [lactate]i by 20 % in soleus and by 26 % in EDL. 5. BZ (0.01 mM) and AZ (0.1 mM), which inhibit only the extracellular sarcolemmal CA, led to a significant increase in dpHs/dt and pHs by about 40 %-150 % in soleus and EDL. BZ and AZ inhibited the influx and efflux of lactate by 25 %-50 % and reduced [lactate]i by about 40 %. The membrane-permeable CA inhibitors CZ (0.5 mM) and EZ (0.1 mM), which inhibit the extracellular as well as the intracellular CAs, exerted no greater effects than the poorly permeable inhibitors BZ and AZ did. 6. In soleus, 10 mM cinnamate inhibited the lactate influx by 47 %. Addition of 0.01 mM BZ led to a further inhibition by only 10 %. BZ alone reduced the influx by 37 %. 7. BZ (0.01 mM) had no influence on the Km value of the lactate transport, but led to a decrease in maximal transport rate (Vmax). In EDL, BZ reduced Vmax by 50 % and in soleus by about 25 %. 8. We conclude that the extracellular sarcolemmal CA plays an important role in lactic acid transport, while internal CA has no effect, a difference most likely attributable to the high internal vs. low extracellular BF(non-HCO3). The fact that the effects of cinnamate and BZ are not additive indicates that the two inhibitors act at distinct sites on the same transport pathway for lactic acid.

Shaking up glycolysis: Sustained, high lactate flux during aerobic rattling

Substantial ATP supply by glycolysis is thought to reflect cellular anoxia in vertebrate muscle. An alternative hypothesis is that the lactate generated during contraction reflects sustained glycolytic ATP supply under well-oxygenated conditions. We distinguished these hypotheses by comparing intracellular glycolysis during anoxia to lactate efflux from muscle during sustained, aerobic contractions. We examined the tailshaker muscle of the rattlesnake because of its uniform cell properties, exclusive blood circulation, and ability to sustain rattling for prolonged periods. Here we show that glycolysis is independent of the O 2 level and supplies one-third of the high ATP demands of sustained tailshaking. Fatigue is avoided by rapid H + and lactate efflux resulting from blood flow rates that are among the highest reported for vertebrate muscle. These results reject the hypothesis that glycolysis necessarily reflects cellular anoxia. Instead, they demonstrate that glycolysis can provide a high and sustainable supply of ATP along with oxidative phosphorylation without muscle fatigue.

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle.

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.

Relationship of MTT reduction to stimulants of muscle metabolism

MTT, a positively charged tetrazolium salt, is widely used as an indicator of cell viability and metabolism and has potential for histochemical identification of tissue regions of hypermetabolism. In the present study, MTT was infused in the constant-flow perfused rat hindlimb to assess the effect of various agents and particularly vasoconstrictors that increase muscle metabolism. Reduction of MTT to the insoluble formazan in muscles assessed at the end of experiments was linear over a 30 min period and production rates were greater in red fibre types than white fibre types. The vasoconstrictors, norepinephrine (100 nM) and angiotensin (10 nM) decreased MTT formazan production in all muscles but increased hindlimb oxygen uptake and lactate efflux. Veratridine, a Na + channel opener that increases hindlimb oxygen uptake and lactate efflux without increases in perfusion pressure, also decreased MTT formazan production. Membrane stabilizing doses (100 μM) of (±)-propranolol reversed the inhibitory effects of angiotensin and veratridine on MTT formazan production. Muscle contractions elicited by stimulation of the sciatic nerve, reversed the norepinephrine-mediated inhibitory effects on MTT formazan production, even though oxygen consumption and lactate efflux were further stimulated. Stimulation of hindlimb muscle oxygen uptake by pentachlorophenol, a mitochondrial uncoupler, was not associated with alterations in MTT formazan production. It is concluded that apart from muscle contractions MTT formazan production does not increase with increased muscle metabolism. Since the vasoconstrictors angiotensin and norepinephrine as well as veratridine activate Na + channels and the Na +/K + pump, energy required for Na + pumping may be required for MTT reduction. It is unlikely that vasoconstrictors that stimulate oxygen uptake do so by uncoupling respiration.

The low-affinity monocarboxylate transporter MCT4 is adapted to the export of lactate in highly glycolytic cells

Transport of lactate and other monocarboxylates in mammalian cells is mediated by a family of transporters, designated monocarboxylate transporters (MCTs). The MCT4 member of this family has recently been identified as the major isoform of white muscle cells, mediating lactate efflux out of glycolytically active myocytes [Wilson, Jackson, Heddle, Price, Pilegaard, Juel, Bonen, Montgomery, Hutter and Halestrap (1998) J. Biol. Chem. 273, 15920-15926]. To analyse the functional properties of this transporter, rat MCT4 was expressed in Xenopus laevis oocytes and transport activity was monitored by flux measurements with radioactive tracers and by changes of the cytosolic pH using pH-sensitive microelectrodes. Similar to other members of this family, monocarboxylate transport via MCT4 is accompanied by the transport of H(+) across the plasma membrane. Uptake of lactate strongly increased with decreasing extracellular pH, which resulted from a concomitant drop in the K(m) value. MCT4 could be distinguished from the other isoforms mainly in two respects. First, MCT4 is a low-affinity MCT: for L-lactate K(m) values of 17+/-3 mM (pH-electrode) and 34+/-5 mM (flux measurements with L-[U-(14)C]lactate) were determined. Secondly, lactate is the preferred substrate of MCT4. K(m) values of other monocarboxylates were either similar to the K(m) value for lactate (pyruvate, 2-oxoisohexanoate, 2-oxoisopentanoate, acetoacetate) or displayed much lower affinity for the transporter (beta-hydroxybutyrate and short-chain fatty acids). Under physiological conditions, rat MCT will therefore preferentially transport lactate. Monocarboxylate transport via MCT4 could be competitively inhibited by alpha-cyano-4-hydroxycinnamate, phloretin and partly by 4, 4'-di-isothiocyanostilbene-2,2'-disulphonic acid. Similar to MCT1, monocarboxylate transport via MCT4 was sensitive to inhibition by the thiol reagent p-chloromercuribenzoesulphonic acid.

Niacin protects the isolated heart from ischemia-reperfusion injury.

Nicotinic acid (niacin) has been shown to decrease myocyte injury. Because interventions that lower the cytosolic NADH/NAD(+) ratio improve glycolysis and limit infarct size, we hypothesized that 1) niacin, as a precursor of NAD(+), would lower the NADH/NAD(+) ratio, increase glycolysis, and limit ischemic injury and 2) these cardioprotective benefits of niacin would be limited in conditions that block lactate removal. Isolated rat hearts were perfused without (Ctl) or with 1 microM niacin (Nia) and subjected to 30 min of low-flow ischemia (10% of baseline flow, LF) and reperfusion. To examine the effects of limiting lactate efflux, experiments were performed with 1) Ctl and Nia groups subjected to zero-flow ischemia and 2) the Nia group treated with the lactate-H(+) cotransport inhibitor alpha-cyano-4-hydroxycinnamate under LF conditions. Measured variables included ATP, pH, cardiac function, tissue lactate-to-pyruvate ratio (reflecting NADH/NAD(+)), lactate efflux rate, and creatine kinase release. The lactate-to-pyruvate ratio was reduced by more than twofold in Nia-LF hearts during baseline and ischemic conditions (P < 0.001 and P < 0.01, respectively), with concurrent lower creatine kinase release than Ctl hearts (P < 0.05). Nia-LF hearts had significantly greater lactate release during ischemia (P < 0.05 vs. Ctl hearts) as well as higher functional recovery and a relative preservation of high-energy phosphates. Inhibiting lactate efflux with alpha-cyano-4-hydroxycinnamate and blocking lactate washout with zero flow negated some of the beneficial effects of niacin. During LF, niacin lowered the cytosolic redox state and increased lactate efflux, consistent with redox regulation of glycolysis. Niacin significantly improved functional and metabolic parameters under these conditions, providing additional rationale for use of niacin as a therapeutic agent in patients with ischemic heart disease.

Transporter reversal as a mechanism of glutamate release from the ischemic rat cerebral cortex: studies with dl- threo-β-benzyloxyaspartate

Elevated levels of the excitotoxic amino acids, glutamate and aspartate, have been implicated in the pathogenesis of neuronal injury and death induced by cerebral ischemia. This study evaluated the contribution of reversed high-affinity, Na +-dependent, glutamate transport to the ischemia-evoked release of glutamate and aspartate using dl- threo-β-benzyloxyaspartate ( dl-TBOA), a newly developed competitive, non-transported blocker of the EAAT 1–3 transporters. Changes in the extracellular levels of these and other amino acids, and of glucose and lactate in cerebral cortical superfusates during four-vessel occlusion-elicited global cerebral ischemia were examined using a cortical window technique. Basal and ischemia-evoked amino acid, glucose and lactate efflux were compared in control versus dl-TBOA (100 μM; applied topically for 35 min prior to ischemia) animals. Twenty minutes of ischemia caused large increases in aspartate, glutamate, GABA and taurine effluxes into cortical superfusates, with non-significant effects on the efflux of glycine, glutamine, alanine and serine. Application of dl-TBOA caused a 2-fold increase in basal, preischemic, extracellular glutamate levels, but did not affect those of the other compounds. In the presence of dl-TBOA, ischemia-evoked release of aspartate, glutamate, taurine and glutamine was significantly reduced; that of the other amino acids was not affected. The ischemia-evoked declines in glucose were significantly attenuated, and lactate release was enhanced above that in control animals. The amino acid data are interpreted as indicating that aspartate and glutamate releases were reduced as a consequence of dl-TBOA inhibition of reversed transport by high-affinity, Na-dependent carriers, predominantly involving the glial EAAT 2 transporter. The reduction in ischemia-evoked taurine release is interpreted as being due to a decrease in cell swelling prior to and during the initial phase of ischemia due to reduced entry of the Na +, and other ions, associated with a decreased glutamate uptake. Glucose-sparing and availability for lactate formation would also result from a reduced glutamate/Na + uptake. These results indicate that reversed transport, primarily from glial cells by the EAAT 2 carrier, is responsible for a substantial (42 and 56%) portion of the ischemia-evoked increase in extracellular glutamate and aspartate levels, respectively. As a potent, competitive, non-transported blocker of high-affinity, Na +-dependent, glutamate transporters, dl-TBOA promises to be a valuable new compound for the study of glutamatergic mechanisms.

Sensitivity of Mechanical and Metabolic Functions to Changes in Coronary Perfusion: A Metabolic Basis of Perfusion–Contraction Coupling

Experimental evidence indicates a metabolic basis of contraction–perfusion coupling during an increase in cardiac work load. This study aims to characterize adjustment of myocardial energy metabolism in response to acute low flow ischemia (LFI), and to determine its involvement in perfusion–contraction coupling. Intracellular parameters were measured in isolated rat hearts by NMR spectroscopy and biochemical methods during 30 min of graded LFI and reperfusion as compared to continuous perfusion (control). Oxygen pressure was set to reach maximal oxygen extraction at 70% coronary flow rate (CFR), therefore oxygen limitation was proportional to coronary underperfusion. At 69, 38 and 10% CFR left ventricular pressures decreased to 71, 43 and 25% of pre-ischemic values respectively (P<0.005 v 97% in control) without an increase in diastolic tone, and recovered to 92±3% after 30 min of reperfusion. Despite hydrolysis of high energy phosphates and cellular acidification, ADP concentrations were stable in underperfused hearts. At 69, 38 and 10% CFR, cytosolic phosphorylation potentials (PP) decreased from 74±10 m M−1during pre-ischemia to 40±6, 25±4 and 14±4 m M−1respectively (P<0.05 v 63±9 m M−1in control), and lactate efflux increased to 256±18, 386±22 and 490±43 μ mol/gdw respectively (P<0.005 v 186±22 μ mol/gdw in control). Glycogen contents decreased (P<0.005 v control) and accounted for 27–30% of lactate efflux. These results indicate: (a) proportionate depression of contraction force and glycogen contents, and increased glucose uptake and anaerobic energy production in the underperfused myocardium. Coordinated modulation of these parameters attributes cytosolic PP a regulatory function; (b) resetting of cytosolic PP to lower levels mediates perfusion–contraction coupling during graded LFI. The data are consistent with the concept that glycolytic energy production improves myocardial tolerance to ischemia.

Uptake of lactate by the liver: effect of red blood cell carriage.

Multiple-indicator dilution experiments with labeled lactate were performed in the livers of anesthetized dogs. A mixture of (51)Cr-labeled erythrocytes, [(3)H]sucrose, and L-[1-(14)C]lactate or a mixture of (51)Cr-labeled erythrocytes, [(14)C]sucrose, and L-[2-(3)H]lactate was injected into the portal vein, and samples were obtained from the hepatic vein. Data were evaluated using a model comprising flow along sinusoids, exchange of lactate between plasma and erythrocytes and between plasma and hepatocytes, and, in the case of L-[1-(14)C]lactate, metabolism to H[(14)C]O(-)(3) within hepatocytes. The coefficient for lactate efflux from erythrocytes was 0.62 +/- 0.24 s(-1), and those for influx into and efflux from hepatocytes were 0.44 +/- 0.13 and 0.14 +/- 0.07 s(-1), respectively. The influx permeability-surface area product of the hepatocyte membrane for lactate (P(in)S, in ml x s(-1) x g(-1)) varied with total flow rate (F, in ml s(-1) x g(-1)) according to P(in)S = (3.1 +/- 0.5)F + (0.021 +/- 0.014). Lactate in plasma, erythrocytes, and hepatocytes was close to equilibrium, whereas lactate metabolism was rate limiting.

Lactate transporters (MCT proteins) in heart and skeletal muscles.

Lactate traverses the cell membranes of many tissues, including the heart and skeletal muscle via a facilitated monocarboxylate transport system that functions as a proton symport and is stereoselective for L-lactate. In the past few years, seven monocarboxylate transporters have been cloned. Monocarboxylate transporters are ubiquitously distributed among many tissues, and the transcripts of several monocarboxylate transporters are present within many of the same tissues. This complicates the identification of their metabolic function. There is also evidence that that there is some species specificity, with differences in MCT tissue distributions in hamsters, rats, and humans. MCT1 and MCT3-M/MCT4 are present in rat and human muscles, and MCT1 expression is highly correlated with the oxidative capacity of skeletal muscles and with their capacity to take up lactate from the circulation. MCT1 is also present in heart and is located on the plasma membrane (in subdomains), T-tubules, and in caveolae. With training, MCT1 is increased in rat and human muscle, and in rat hearts, resulting in an increased uptake of lactate from the buffers perfused through these tissues and an increase in lactate efflux out of purified vesicles. In humans, the training-induced increases in MCT1 are associated with an increased lactate efflux out of muscle. MCT3-M/MCT4 is not correlated with the muscles' oxidative capacities but is equally abundant in Type IIa and IIb muscles, whereas it is markedly lower in slow-twitch (Type I) muscles. Clearly, we are at the threshold of a new era in understanding the regulation of lactate movement into and out of skeletal muscle and cardiac cells.

What governs skeletal muscle VO2max? New evidence.

Recent investigations into the determinants of skeletal muscle maximal oxygen consumption (VO2) have provided further evidence regarding the role of O2 supply and demand in governing exercise metabolism. Specifically, four studies utilizing both animal and human exercise models are highlighted here: 1) the role of the diffusive O2 component was examined in the exercising canine gastrocnemius muscle by a rightward shift in the O2 dissociation curve while maintaining O2 delivery constant; 2) the role of peripheral and central components was examined by studying the human quadriceps muscle, already recognized to have a very high mass specific O2 delivery, under conditions of increased (hyperoxia) and reduced O2 availability (hypoxia); 3) the role of intracellular PO2 in the progressive increase in lactate efflux from skeletal muscle from submaximal to maximal effort; and finally 4) the role of intracellular PO2 itself as a determinant of maximal mitochondrial O2 consumption. In summary, these investigations illustrate 1) the importance of the diffusion gradient from blood to muscle cell; 2) illustrate that even in functionally isolated trained skeletal muscle the highest recorded metabolic rates can be increased by increasing O2 supply; 3) that a constant intracellular PO2 during graded exercise is therefore unrelated to increasing lactate efflux; and 4) that only in hyperoxia does trained human skeletal muscle approaching very high mitochondrial metabolic limits, as shown by a disproportionate increase in intracellular PO2 for the recorded change in VO2max.

A Monocarboxylate Transporter MCT1 is Located at the Basolateral Pole of Rat Jejunum

We have functionally expressed and identified a monocarboxylate transporter (MCT1) from rat jejunal enterocyte and we provide evidence for its basolateral localization. Poly(A)+ RNA isolated from rat jejunum was injected into Xenopus laevis oocytes and expression of a proton-lactate symporter was investigated by means of L-[14C]lactate uptake. The existence of an endogenous capacity for L-lactate transport was demonstrated; when, however, oocytes were injected with jejunal mRNA, an expressed L-lactate uptake was seen which differed from the endogenous transporter since it was significantly pH dependent. After sucrose density gradient fractionation, the highest expression of the pH-dependent lactate uptake was detected with the mRNA size fraction of about 2-3 kb in length. The substrate specificity, stereoselectivity and sensitivity to pCMBS (an organomercurial thiol reagent that modifies cysteine residues) of the expressed transport were in good agreement with results previously obtained using isolated jejunal basolateral membranes. Using the reverse transcriptase-polymerase chain reaction, the presence of mRNA coding for the MCT1 isoform was demonstrated in jejunal enterocytes. These data, together with previous results, suggest that MCT1 is a major route for lactate efflux across the basolateral membrane of rat jejunum; this is in contrast to current opinion which restricts the presence of MCT1 to the apical membrane of the whole small intestine.

Na+channel and Na+-K+ATPase involvement in norepinephrine- and veratridine-stimulated metabolism in perfused rat hind limb

In the constant flow perfused rat hind limb, norepinephrine (NE) evoked increases in oxygen uptake (VO2) and lactate efflux (LE) were inhibited by the cardiac glycoside ouabain (1 mM), without interrupting the NE-mediated vasoconstriction. The membrane labilizer veratridine, previously shown to increase VO2 and LE, without increasing perfusion pressure, was also shown to be inhibited by the cardiac glycoside ouabain, as well as by the ouabain analogues digitoxin and digoxin. The stimulatory actions of veratridine on VO2 were inhibitable by low doses of the specific sodium channel blocker tetrodotoxin (TTX), while NE effects were unaffected, suggesting that NE may be acting via a TTX-insensitive sodium channel. It is concluded that agents such as NE (a vasoconstrictor) or veratridine (a membrane labilizer), which stimulate VO2 in the perfused rat hind limb, do so by increasing Na+ influx. The observed increases in oxygen consumption and LE are due to Na+-K+ ATPase activity to pump Na+ out of the cell at the expense of ATP turnover. Energy dissipation due to Na+ cycling may be a form of facultative thermogenesis attributable to NE that can be stimulated by membrane labilizers such as veratridine in the constant flow perfused rat hind limb.

Cerebral oxygen/glucose ratio is low during sensory stimulation and rises above normal during recovery: excess glucose consumption during stimulation is not accounted for by lactate efflux from or accumulation in brain tissue.

Functional activation stimulates CMRglc more than CMRO2 and raises lactate levels in brain. This has been interpreted as evidence that brain work is supported mainly by energy derived from anaerobic glycolysis. To determine if lactate production accounts for the "excess" glucose consumption, cerebral arteriovenous differences were measured in conscious rats before, during, and 15 minutes after sensory stimulation; the brains were rapidly frozen in situ immediately after completion of blood sampling and assayed for metabolite levels. The molar O2/glucose uptake ratio fell from 6.1+/-1.1 (mean+/-SD) before stimulation to 5.0+/-1.1 during activation (P<0.01); lactate efflux from brain to blood was detectable at rest but not during stimulation. By 15 minutes after activation, O2 and lactate arteriovenous differences normalized, whereas that for glucose fell, causing the O2/glucose ratio to rise above preactivation levels to 7.7+/-2.6 (P<0.01). Brain glucose levels remained stable through all stages of activity. Brain lactate levels nearly doubled during stimulation but normalized within 15 minutes of recovery. Brain glycogen content fell during activation and declined further during recovery. These results indicate that brain glucose metabolism is not in a steady state during and shortly after activation. Furthermore, efflux from and increased content of lactate in the brain tissue accounted for less than 54% of the "excess" glucose used during stimulation, indicating that a shift to anaerobic glycolysis does not fully explain the disproportionately greater increases in CMRglc above that of CMRO2 in functionally activated brain. These results also suggest that the apparent dissociation between glucose utilization and O2 consumption during functional activation reflects only a temporal displacement; during activation, glycolysis increases more than oxidative metabolism, leading to accumulation of products in intermediary metabolic pools that are subsequently consumed and oxidized during recovery.